Project Summary Vitamin C is an indispensable micronutrient for normal human health and well-being. Vitamin C deficiency leads to a variety of clinical abnormalities. The vitamin acts as a potent antioxidant and a cofactor for several enzymes, with low intracellular levels causing oxidative stress, a driver for many human diseases. Therefore, studies designed to optimize overall vitamin C body homeostasis are important. Humans have lost the ability to synthesis vitamin C endogenously, and must obtain it via intestinal absorption. The intestinal absorption process involves the human sodium-dependent vitamin C transporters-1 & 2 (hSVCT1& hSVCT2), where hSVCT1 is exclusively expressed at the apical membrane of the polarized enterocytes whereas hSVCT2 is localized basolaterally. The objectives of this proposal are to continue our investigations into the molecular physiology/cell biology of intestinal vitamin C uptake process, and to address specific aspects of its pathophysiology as well as to determine the effect of external/internal factors on the uptake process. Our new preliminary studies suggest the involvement of microRNA and epigenetic mechanism(s) in the regulation of hSVCT1 expression, identified putative novel hSVCT1 interacting partners, and show a significant inhibition in vitamin C uptake upon exposure to specific enteric pathogens (EPEC and ETEC), pro-inflammatory cytokines, and to bacterial LPS. Based on these findings, our working hypotheses are: i) microRNA and epigenetic mechanism(s) regulate SVCT1 expression and function; ii) hSVCT1 has interacting partners that affect its physiology/cell biology; and iii) exposure to enteric pathogens, pro- inflammatory cytokines, and to bacterial LPS leads to a significant inhibition in intestinal vitamin C uptake. Three specific aims are proposed to test these hypotheses and will utilize state-of- the-art cell/molecular approaches. Results of these investigations should provide valuable information regarding the intestinal vitamin C absorption process under normal physiological conditions, and how this event is affected by specific pathophysiological factors. This should ultimately help us in designing effective strategies to optimize normal vitamin C body homeostasis, especially in conditions of deficiency/sub-optimal levels.